GSM6589521: SRSF10_ LACEseq_rep2; Mus musculus; RNA-Seq
The samples were firdtly irradiated twice with UV-Clight on ice at 400mJ. 10 µl of protein A/G magnetic beads (per sample, Thermo Scientific, 26162) were washed twice with BSA/PBS solution (0.1% BSA in 1 × PBS) and incubated with 200 µl blocking buffer (1 × PBS, 0.2 mg/ml glycogen, 0.2 mg/ml BSA) at room temperature (RT) for 1 h. The blocked beads were then washed once with 0.1 M Na-phosphate buffer (93.2 mM Na2HPO4, 6.8 mM NaH2PO4, 0.05% tween 20, pH 8.0) and then resuspended in 40 µl of 0.1 M Na-phosphate buffer containing 5 µg SRSF10 antibody (Cat No.) or IgG (Cat No.), incubating at RT for 1 h. The antibody-coupled beads were washed twice with wash buffer (1 × PBS, 0.1% SDS, 0.5% NP-40, 0.5% sodium deoxycholate) and resuspended in 10 µl of wash buffer per sample. The cross-linked samples were lysed on ice with 50 µl of wash buffer for 10 min. After removal of genomic DNA with 4 µl of RQ1 DNase (Promega, M6101) in the presence of 1µl of RNase inhibitor (Thermo Scientific, EO0381), 10 µl of antibody-coupled beads were added to the lysate and incubated for 1 h at 4 °C. Bead-bound antibody-RNA complexes were then washed twice with wash buffer, once with high-salt wash buffer (5 × PBS, 0.1% SDS, 0.5% NP-40, 0.5% sodium deoxycholate), and once with PNK buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.5% NP-40). The immunoprecipitated RNAs were then fragmented with 1×10-5 U of micrococcal nuclease (MNase, New England BioLabs, M0247S) in 1 × MN reaction buffer (50 mM Tris-HCl, pH 8.0, 5 mM CaCl2) for 3 min at 37 °C. After another round of extensive washing (twice with 1 × PNK + EGTA buffer (50 mM Tris-HCl, pH 7.4, 20 mM EGTA, 0.5% NP-40), twice with wash buffer, and twice with PNK buffer), the RNA 3' ends were dephosphorylated on beads with FastAP alkaline phosphatase (Thermo Scientific, EF0651) for 15 min in FastAP buffer. After washing beads twice with 1 × PNK + EGTA buffer, twice with 1 × PNK buffer, and twice with BSA solution (0.2 mg/ml BSA in DEPC water), the 3' linker was ligated with T4 RNA ligase 2 (New England BioLabs, M0242) for 2.5 h at RT. After washing beads three times with 1 × PNK buffer, the RNA was reverse transcribed with Superscript II reverse transcriptase (Thermo Scientific,18064014) according to the manufacturer's instruction. Then excess T7-RT primer was digested with Exonuclease I (New England BioLabs, M0293), first-strand cDNA was released from Protein A/G beads by treatment with RNase H (Thermo Scientific, EN0202) and captured by streptavidin C1 beads (Thermo Scientific, 650002). After washing beads twice with 1 × B & W buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl), once with BSA solution, the cDNA 3' linker was ligated with T4 RNA ligase 1 (New England BioLabs, M0437) overnight at RT. The cDNA was pre-amplified using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KK2601), and PCR products were purified with Ampure XP beads (Beckman Coulter, A63881). Then in vitro transcription was performed, the DNA template was removed with TURBO DNase (Thermo Scientific, AM2238) and the RNA was purified with Agencourt RNA Clean beads (Beckman Coulter, A63987). After reverse transcription and indexed PCR, the PCR products were size-selected on a 2% agarose gel, and regions corresponding to 250–500 bp were purified using Gel Extraction Kit (Qiagen, 28604). The LACE-seq library was paired-end sequenced using Illumina NovaSeq 6000 at Novogene.